N-acetylhexosamine-containing n-glycans in glycoprotein products

ABSTRACT

The present invention provides methods of evaluating a glycoprotein preparation for the absence, presence or amount of an N-acetylhexosamine glycan, e.g., an N-acetylglucosamine glycan.

This application claims priority to U.S. Application Ser. No.61/452,092, filed on Mar. 12, 2011. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

FIELD OF THE INVENTION

The present invention relates to methods and materials for the detectionof particular glycan structures in glycoproteins.

BACKGROUND OF THE INVENTION

Antibodies represent a growing class of biotherapeutic drugs in thepharmaceutical market and in development for a variety of indications.Unlike a small molecules, biologics are actually a mixture of isoformsall of which are contain the same peptide backbone, but differ inmodifications and which may have a range of pharmacokinetic,pharmacodymanic or safety profiles. It is therefore important toroutinely monitor product quality. Antibodies are glycoproteins whichcontain at least one variably occupied N-glycosylation site.N-glycosylation in antibody therapeutics is thought to influencepharmacokinetics and structural integrity of the molecule (Krapp, Mimuraet al. 2003).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of anN-acetylhexosamine glycan structure that can be present onglycoproteins, e.g., a glycosylated antibody. In some embodiments, thepresence or amount of the N-acetylhexosamine is associated with aparticular production parameter or parameters. The absence, presenceand/or amount of this glycan structure can be used, inter alia, forevaluating or processing a glycoprotein preparation, e.g., to determinewhether to accept or reject a batch of a glycoprotein, e.g., aglycosylated antibody, or to guide or control a production parameter orparameters used to produce a glycoprotein, e.g., a glycosylatedantibody. In some embodiments, the absence, presence or amount of thisstructure in a glycoprotein composition can be compared, e.g., to areference standard, e.g., to make a decision regarding the glycoproteinpreparation, e.g., a decision to classify, select, accept or discard,release or withhold, process into a drug product, ship, move to adifferent location, formulate, label, package, release into commerce, orsell or offer for sale the glycoprotein, e.g., a glycosylated antibody.In other embodiments, the decision can be to accept, modify or reject aproduction parameter or parameters used to make the glycoprotein, e.g.,a glycosylated antibody.

Thus, in a first aspect, the disclosure features methods for evaluatinga glycoprotein preparation. In some embodiments, the method includes:

-   -   providing one or more preparations of a glycoprotein, e.g., a        glycosylated antibody preparation; and    -   determining, e.g., using a separation method, the absence,        presence and/or amount of an N-acetylhexosamine glycan        associated with the glycoprotein.

The determining step may include one or more of the following: (a)isolating a glycoprotein produced by a cell and determining if anN-acetylhexosamine glycan is present on the glycoprotein, (b) isolatinga glycoprotein preparation produced by a cell and determining thepresence or amount of the N-acetylhexosamine on glycoproteins of thepreparation, (c) isolating glycans from a glycoprotein produced by acell and determining if the glycans of the glycoprotein include anN-acetylhexosamine glycan, and (d) providing at least one peptide from aglycoprotein produced by cell, and determining the presence glycanscontaining an N-acetylhexosamine on the at least one peptide. Thetechnique used to measure the N-acetylhexosamine can include one or moreof the following methods, and combinations of any of these methods:chromatographic methods, mass spectrometry (MS) methods, electrophoreticmethods (such as capillary electrophoresis), nuclear magnetic resonance(NMR) methods, monosaccharide analysis, fluorescence methods, UV-VISabsorbance, enzymatic methods, and use of a detection molecule (such asan antibody or lectin).

The source of glycans can be selected from the group consisting of: apopulation of cells, e.g., CHO cells; an isolated glycoprotein, e.g., anisolated glycosylated antibody; peptides derived from the cleavage of aglycoprotein, e.g., a glycosylated antibody; or glycans derived from theglycoprotein. In some embodiments, the method includes treating a sourceof glycans or glycopeptides with one or more enzymes, e.g., PNGase,followed by analysis of the glycan population. In some embodiments thismethod includes treating the glycopeptides with a chemical to releasethe glycan, e.g. acid hydrolysis, followed by analysis of the releasedglycans or monosaccharides and or analysis of the glycan attached to asingle amino acid.

In some embodiments, the method used provides a quantitative measure ofan N-acetylhexosamine glycan. In some embodiments, the method usedprovides a qualitative measure.

In some embodiments, the method also includes preparing a glycoproteinpreparation, cleaving one or more glycans from the glycoproteinpreparation (e.g., with one or more enzyme such as PNGase, anddetermining the absence, presence or amount of an N-acetylhexosamineglycan.

In certain embodiments, the method is conducted during a production runfor a therapeutic glycoprotein by obtaining a sample from the cellculture of the production line, e.g., to monitor glycan structure duringproduction. In certain embodiments, the determining step is repeated atleast once over time, e.g., the determining step is repeated at leastonce, twice, three times or more, during the time period of culture ofthe cells. In some embodiments, the method is conducted during thestorage of a glycoprotein by obtaining a sample from the glycoproteincomposition, e.g., to monitor glycan structure stability during storage.In certain embodiments, the determining step is repeated at least onceover time, e.g., the determining step is repeated at least once, twice,three times or more, during the storage of the glycoprotein composition.In other embodiments, the method is conducted on a glycoproteincomposition, e.g., as part of a quality or release testing of theglycoprotein composition.

In some embodiments, the determining step includes comparing the levelof N-acetylhexosamine glycan containing glycoproteins in a firstglycoprotein preparation produced from a first population of cells,e.g., produced under a first production parameter or parameters, to thelevel of N-acetylhexosamine glycan containing glycoprotein in a secondglycoprotein preparation produced from a second population of cellsand/or the same cells under a different production parameter orparameters. In some such embodiments, the presence or amount of anN-acetylhexosamine glycan of a glycoprotein preparation is determinedand compared to the presence or amount of the N-acetylhexosamine glycanof a glycoprotein produced under a different production parameter orparameters.

In some embodiments, the method comprise a step of comparing the levelof N-acetylhexosamine glycans to a reference standard (e.g., to acontrol level, or to a range or value in a product specification).

In certain embodiments of the method, the determining step includes useof a detection molecule which is able to detect the presence or absenceof an N-acetylhexosamine glycan. In certain embodiments, the detectionmolecule comprises an antibody that is able to bind toN-acetylhexosamine. In some embodiments, the detection molecule maycomprise a fluorescent moiety, or a radioisotope moiety. In someembodiments this comprises another sugar that forms a covalent linkageto the N-acetylhexosamine. In some embodiments, the detection moleculemay comprise a fluorescent moiety, or a radioisotope moiety. In someembodiments this comprises another sugar that forms a covalent linkageto the N-acetylhexosamine. In some embodiments, the detection moleculemay comprise a fluorescent moiety, or a radioisotope moiety.

In some embodiments, the glycoprotein, e.g., the glycosylated antibody,is produced by a clonal cell population, e.g., a clonal CHO cellpopulation. The cell population may be in culture, e.g., or a samplefrom a cell culture in a bioreactor for manufacturing the glycoprotein,e.g., the glycosylated antibody. In certain embodiments, the cellpopulation will have been transformed with at least one vector encodinga glycoprotein. The therapeutic glycoprotein may be of human, non-humanor synthetic origins. In some embodiments, the glycoprotein may be fortreatment of humans or veterinary indications.

In some embodiments, the method further includes a step of evaluating abiological activity of the glycoprotein produced by the cell, e.g.,evaluating the presence or level of immunogenic potential of theglycoprotein, e.g., in vitro or in vivo, e.g., in an animal model.

In a second aspect, the invention comprises methods for screening one ormore cells for the ability to produce an N-acetylhexosamine glycan on aglycoprotein, the method comprising:

-   -   providing a plurality of cell populations, e.g., a plurality of        CHO cell populations; culturing each of the plurality of cells        under conditions suitable for expression of a glycoprotein;    -   measuring N-acetylhexosamine glycans (e.g., N-acetylglucosamine        and/or N-acetylgalactosamine) produced by each of the plurality        of cells, and    -   selecting one or more of the plurality of cell preparations        based on the presence of a target level of N-acetylhexosamine        glycans produced by the selected cell preparation.

In some embodiments, the cell population is a CHO cell population.

The N-acetylhexosamine glycans can be obtained and measured fromglycoproteins produced by the cell preparations, from an isolatedglycoprotein of the cell preparations, from peptides obtained from aglycoprotein produced by the cell preparations, or from glycanpreparations obtained from the cell preparations or from a glycoproteinproduct thereof. In certain embodiments, the screening method furthercomprises the step of isolating a glycoprotein expression product fromthe cell culture and measuring for the presence or amount of anN-acetylhexosamine glycan on a glycoprotein produced by the cells instep (c). In certain embodiments, the cell screening method comprisesquantifying the amount of N-acetylhexosamine glycans present on theglycoprotein preparation. In certain embodiments, step (b) of the cellscreening method takes place in a bioreactor.

Each of the plurality of cell populations may comprise a differentstrain population, a different clonal cell population, or differentsamples (e.g., samples taken over time) from a cell culture used tomanufacture a glycoprotein. In certain embodiments, the cell populationis transformed with at least one vector encoding a glycoprotein, e.g., aglycosylated antibody, e.g., a human or humanized glycosylated antibody.In certain embodiments of the cell screening method, the glycoprotein isa secreted glycoprotein expressed from the cells.

The measuring step of the screening method may include any techniquedisclosed herein for identifying and/or quantifying anN-acetylhexosamine glycan on the glycoprotein.

In a third aspect, the invention includes a method for evaluating aglycoprotein preparation. The method includes measuring the amount of anN-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/orN-acetylgalactosamine glycan) in a glycoprotein preparation, e.g., aglycosylated antibody preparation.

In some embodiments, the glycoprotein preparation is produced in a hostcell, e.g., a prokaryotic or eukaryotic host cell. The eukaryotic cellcan be, e.g., a yeast, an insect, a fungi, a plant or an animal cell(e.g., a mammalian cell). Exemplary host cells are described herein.

In some embodiment, the method includes recording the absence, presenceor amount of N-aceytlhexosamine glycans in the glycoprotein preparationin a print or computer-readable medium.

In some embodiments, the method also includes comparing the measuredlevel of N-acetylhexosamine glycan present in the glycoproteinpreparation with a reference standard, such as a control or referencespecification. The reference standard can be a specification (e.g., anFDA label or Physician's Insert) or quality criterion for apharmaceutical preparation containing the glycoprotein preparation.

In some embodiment, the level of N-acetylhexosamine glycans present in aglycoprotein preparation can be measured as the level ofN-acetylhexosamine glycans relative to total amount of glycans in asample, such as a glycoprotein preparation.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes a chromatographic method.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes mass spectrometry (MS) methods.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes electrophoretic methods (such as capillaryelectrophoresis).

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes nuclear magnetic resonance (NMR) methods.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes monosaccharide analysis.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes fluorescence methods.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes UV-VIS absorbance.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes enzymatic methods.

In one embodiment, the technique used to measure N-acetylhexosamineglycan content includes and use of a detection molecule (such as anantibody).

In another aspect, the invention includes a recombinant glycoproteinthat has a different level of N-acetylhexosamine glycans than areference glycoprotein that has the same or highly similar amino acidsequence. A highly similar amino acid sequence, as used herein, is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, the reference glycoprotein is a commerciallyavailable therapeutic glycoprotein, e.g., a therapeutic glycoproteindisclosed in Table 2. The recombinant glycoprotein can have a higher orlower level of N-acetylhexosamine glycans than the referenceglycoprotein, e.g., the recombinant glycoprotein can have at least 2%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% higher orlower level of N-acetylhexosamine glycans, e.g., as measured as apercent of total glycans, or as measured relative to the total amount ofG0F, G1F, G2F and glycosylated glycopeptides. In one embodiment, therecombinant glycoprotein is an IgG1 preparation, and the recombinantglycoprotein has a level of N-acetylhexosamine glycans greater than orless than 3%, 5%, 10%, or 15% as measured relative to the total amountof G0F, G1F, G2F and glycosylated glycopeptides. In another embodiment,the recombinant glycoprotein is an IgG1 preparation, and the recombinantglycoprotein has a level of N-acetylhexosamine glycans greater than orless than 40%, 50% or 55% as measured relative to the total amount ofG0F, G1F, G2F and glycosylated glycopeptides. In yet another embodiment,the recombinant glycoprotein is an IgG2 preparation, and the recombinantglycoprotein has a level of N-acetylhexosamine glycans greater than orless than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or 25% asmeasured relative to the total amount of G0F, G1F, G2F and glycosylatedglycopeptides.

In another aspect, the disclosure features a method for modulatingeffector function of an antibody preparation. In some embodiments, themethod includes:

providing an antibody preparation; and

modulating, e.g., increasing or decreasing, the N-acetylhexosamineglycan content of the antibody preparation.

In some embodiments, the method comprises increasing N-acetylhexosamineglycan content of the antibody preparation, e.g., to thereby decreaseeffector function of the antibody preparation. In some embodiments, themethod comprises removing one or more glycan structure associated witheffector function (e.g., a sialylated glycan), and e.g., the addition ofan N-acetylhexosamine glycan. In other embodiments, the method comprisesenzymatically or chemically modifying the glycan structure to form anN-acetylhexosamine glycan, e.g., with enzymes or chemicals describedherein.

In other embodiments, the method comprises decreasing N-acetylhexosamineglycan content of the antibody preparation, e.g., to thereby increaseeffector function of the antibody preparation. In some embodiments, themethod comprises removing one or more N-acetylhexosamine glycans, ande.g., the addition of glycan structure associated with effectorfunction, e.g., a sialylated glycan. In other embodiments, the methodcomprises enzymatically or chemically modifying the glycan structure toform a glycan structure associated with increased effector function.

In another aspect, the disclosure features a method for evaluatingeffector function of an antibody preparation. In some embodiments, themethod includes:

-   -   providing an antibody preparation; and    -   determining the absence, presence or amount of        N-aceytlhexosamine glycans in the preparation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of the N-acetylhexosamine glycan structures.

FIG. 2 depicts MS/MS fragmentation of IgG Fc glycopeptides containing asingle N-linked N-acetylhexosamine glycan. The fragmentation patternplaces the modification in the Asp residue, residue 297, in the middleof the sequence.

FIG. 3 is a graph depicting the percentage of truncated N-linkedN-acetylhexosamine glycan present in commercially available IgGglycoproteins. Relative abundance of the N-acetylhexosamine glycancontaining glycoproteins is calculated based upon the abundance of theG0F, G1F, G2F and the aglycosyl glycopeptides. These species comprisemore than 85% of the glycan composition for each of the commerciallyavailable IgGs tested.

DEFINITIONS

Unless otherwise defined herein below, all terms used herein are used intheir ordinary meaning, as would be understood by one skilled in theart.

Approximately, About, Ca.: As used herein, the terms “approximately”,“about” or “ca.,” as applied to one or more values of interest, refer toa value that is similar to a stated reference value. In certainembodiments, the terms “approximately”, “about” or “ca.,” refer to arange of values that fall within 5%, 4%, 3%, 2%, 1%, or less of thestated reference value.

Detection, Detecting: As used herein, the terms “detecting,” “detection”and “detecting means” are used interchangeably to refer to thedetermination of whether a particular chemical moiety, such as anN-acetylhexosamine (e.g., an N-acetylglucosamine and/orN-acetylgalactosamine), is present or absent in or on a compound,preparation, composition, cell or cell population. The detecting meansmay involve a selectable marker, or an identifiable characteristic suchas a fluorescent or radioactive moiety, and may involve labeling of areagent, compound, cell or cell population. Detection can also refer tothe analysis of a compound, preparation, composition, cell or cellpopulation, using such techniques as mass spectrometry or relatedmethods, electrophoretic methods, nuclear magnetic resonance,chromatographic methods, or combinations of the above, to determine thepresence or absence of a chemical moiety in or on a compound,preparation, composition, cell or cell population. Detection may alsoinvolve quantification of the absolute or relevant levels of thechemical moiety being detected.

Glycan: As is known in the art and used herein “glycans” are sugars.Glycans can be monomers or polymers of sugar residues, and can be linearor branched. A glycan may include natural sugar residues (e.g., glucose,N-acetylglucosamine, N-acetylgalactosamine, N-acetyl neuraminic acid,galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.)and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose,phosphomannose, 6′sulfo N-acetylglucosamine, etc.). The term “glycan”includes homo and heteropolymers of sugar residues. The term “glycan”also encompasses a glycan component of a glycoprotein (e.g., of aglycoprotein, glycolipid, proteoglycan, etc.). The term also encompassesfree glycans, including glycans that have been cleaved or otherwisereleased from a glycoprotein.

Glycan preparation: The term “glycan preparation” as used herein refersto a set of glycans obtained according to a particular productionmethod. In some embodiments, glycan preparation refers to a set ofglycans obtained from a glycoprotein preparation (see definition ofglycoprotein preparation below). In some embodiments, a glycanpreparation includes glycoproteins. In some embodiments, a glycanpreparation includes released glycans.

Glycoprotein: As used herein, the term “glycoprotein” refers to a“protein” (as defined herein) that contains a peptide backbonecovalently linked to one or more sugar moieties (i.e., glycans). As isunderstood by those skilled in the art, the peptide backbone typicallycomprises a linear chain of amino acid residues. The sugar moiety(ies)may be in the form of monosaccharides, disaccharides, oligosaccharides,and/or polysaccharides. The sugar moiety(ies) may comprise a singleunbranched chain of sugar residues or may comprise one or more branchedchains. In certain embodiments, sugar moieties may include sulfateand/or phosphate groups. Alternatively or additionally, sugar moietiesmay include acetyl, glycolyl, propyl or other alkyl modifications. Incertain embodiments, glycoproteins contain O-linked sugar moieties; incertain embodiments, glycoproteins contain N-linked sugar moieties.

Glycoprotein preparation: A “glycoprotein preparation,” as that term isused herein, refers to a set of individual protein molecules, each ofwhich comprises a particular amino acid sequence (which amino acidsequence includes at least one glycosylation site) and a plurality ofthe proteins have at least one glycan covalently attached to the atleast one glycosylation site. Individual molecules of a particularglycoprotein within a glycoprotein preparation typically have identicalamino acid sequences but may differ in the occupancy of the at least oneglycosylation sites and/or in the identity of the glycans linked to theat least one glycosylation sites. That is, a glycoprotein preparationmay contain only a single glycoform of a particular glycoprotein, butmore typically contains a plurality of glycoforms. Differentpreparations of the same glycoprotein may differ in the identity ofglycoforms present (e.g., a glycoform that is present in one preparationmay be absent from another) and/or in the relative amounts of differentglycoforms.

Glycoprotein composition: A “glycoprotein composition” as used hereinrefers to a glycoprotein preparation that is in the form of a drugsubstance or drug product.

Glycosidase: The term “glycosidase” as used herein refers to an agentthat cleaves a covalent bond between sequential sugars in a glycan orbetween the sugar and the backbone moiety (e.g., between sugar andpeptide backbone of glycoprotein). In some embodiments, a glycosidase isan enzyme. In certain embodiments, a glycosidase is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a glycosidase is a chemical cleavage agent, e.g.,hydrazine.

N-glycan: The term “N-glycan,” as used herein, refers to a polymer ofsugars that has been released from a glycoprotein but was formerlylinked to a glycoprotein via a nitrogen linkage (see definition ofN-linked glycan below).

N-linked glycans: N-linked glycans are glycans that are linked to aglycoprotein via a nitrogen linkage. A diverse assortment of N-linkedglycans exists, but is typically based on the common corepentasaccharide (Man)₃(GlcNAc)(GlcNAc).

O-glycan: The term “O-glycan,” as used herein, refers to a polymer ofsugars that has been released from a glycoconjugate but was formerlylinked to the glycoconjugate via an oxygen linkage (see definition ofO-linked glycan below).

O-linked glycans: O-linked glycans are glycans that are linked to aglycoconjugate via an oxygen linkage. O-linked glycans are typicallyattached to glycoproteins via N-acetyl-D-galactosamine (GalNAc) or viaN-acetyl-D-glucosamine (GlcNAc) to the hydroxyl group of L-serine (Ser)or L-threonine (Thr). Some O-linked glycans also have modifications suchas acetylation and sulfation.

Modulate: The term “modulate” as used herein refers to the ability of anactor to control, within prescribed limits, the value of a parameter,such as the level of N-acetylhexosamine glycans present in aglycoprotein preparation. Thus, in some embodiments, the level ofN-acetylhexosamine glycans may be modulated so that it remains withinprescribed limits. In some embodiments, the level of N-acetylhexosamineglycans may be modulated so that it does not vary by more than 10.0%,5.0%, 1.0%, 0.5% or 0.1% of a reference standard.

Protease: The term “protease” as used herein refers to an agent thatcleaves a peptide bond between sequential amino acids in a polypeptidechain. In some embodiments, a protease is an enzyme (i.e., a proteolyticenzyme). In certain embodiments, a protease is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a protease is a chemical cleavage agent.

Providing: The term “providing” as used herein refers to an actorobtaining a subject item, such as a cell preparation, or glycoproteinpreparation, from any source including, but not limited to, obtaining bythe actor's own manufacture or by the actor's receiving the item fromanother party. For example, a cell preparation is provided if it is madeor received by any machine, person, or entity. In some embodiments, acell preparation may be received by a machine, which may then performone or more tests, processes, or refinements of the glycoproteinpreparation. In some embodiments, a cell preparation may be received bya person. In some embodiments, a CHO cell preparation may be receivedfrom an outside entity. In some embodiments, a cell preparation may bereceived by a person or business performing characterization servicesfor a second person or business.

N-acetylhexosamine glycan: The term “N-acetylhexosamine glycan” as usedherein, describes the glycan structures illustrated in FIG. 1.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Although host cells used for the synthesis of recombinant glycoproteinspossess the intracellular machinery to produce complex glycosylation,these cells do not always possess the same complement of enzymes as thecells in which the glycoprotein is naturally expressed. Clonal selectionof cell lines and variations in manufacturing conditions may alsoproduce heterogeneity in glycoproteins expressed in cultured cells. Thefunctional role of glycosylation in glycoprotein activity necessitatescareful characterization of therapeutic products produced in cell lines.

The present disclosure is based, at least in part, on the unexpectedfinding that many glycoprotein preparations, e.g., glycosylated antibodypreparations, contain an unusual N-linked N-acetylhexosamine glycanstructure. For example, it has been found that an N-linkedN-acetylhexosamine glycan can be found at the glycosylation site Asn297of the Fc region of antibodies within a glycosylated antibodypreparation. Many glycosylated antibody preparations contain thisstructure and, thus it is important to identify, monitor and controlthis aspect of glycan structure when producing glycosylated antibodypreparations.

The present disclosure provides methods of analyzing the composition ofglycans on glycoproteins. According to the present disclosure, glycansfrom glycoprotein preparations can be analyzed to determine whether theyinclude an N-acetylhexosamine glycan. The present disclosure providesmethods of detecting the absence, presence or amount ofN-acetylhexosamine glycans associated with a glycoprotein preparation,e.g., a glycosylated antibody preparation, e.g., a glycosylated antibodypreparation described herein, and methods of producing glycoproteinsthat include, include is a certain amount or lack this glycan structure.

Glycan Preparations

The present disclosure provides methods of analyzing the structureand/or composition of individual glycans within a glycan preparation,e.g., evaluating for the absence, presence or amount ofN-acetylhexosamine glycans (N-acetylglucosamine and/orN-acetylgalactosamine). A glycan preparation may be obtained from a cellpreparation or from a glycoprotein made by any method available in theart. In general, obtaining a glycan preparation comprises steps of (1)obtaining a cell or glycoprotein preparation; and (2) optionallyreleasing glycans from the cell or glycoprotein preparation. In someembodiments, obtaining a glycan preparation optionally compriseslabeling the glycan preparation with a detectable label.

Glycoprotein Preparations

Methods for recombinant production of glycoproteins have been described.Glycoproteins secreted by cultured cells can be isolated and purified byany available means, such as anion-exchange chromatography,reversed-phase chromatography, gel filtration, immunoaffinitychromatography, and combinations thereof.

N-Linked Glycan Preparation

In some embodiments, an N-glycan preparation is obtained by providing aglycoprotein population and removing N-linked glycans from theglycoproteins in the population.

In some embodiments, N-linked glycans are removed from glycoproteins(e.g., glycoproteins) by digestion. Generally, glycanases to be used inaccordance with the present disclosure cleave between GlcNAc-Asn,GlcNAc-GlcNAc, or Man-GlcNAc residues of the core. Exemplary enzymeswhich can be used to remove N-linked glycans from glycoproteins include,but are not limited to, N-glycanase F and/or N-glycanase-A, O-glycanaseand/or Endo H.

In some embodiments, N-linked glycans are removed from glycoproteins bychemical cleavage. To give but a few examples, hydrazine, sodiumborohydride, and/or trifluoromethanesulfonic acid (TFMS) can be used toremove glycans from a glycoprotein.

O-Linked Glycan Preparation

In some embodiments, an O-linked glycan preparation is obtained byproviding a glycoprotein (e.g., glycoprotein) population and removingO-linked glycans from glycoproteins in the population.

In some embodiments, O-linked glycans are removed from glycoproteins(e.g., glycoproteins) by beta elimination. In some embodiments, O-linkedglycans are removed from glycoproteins (e.g., glycoproteins) byreductive beta elimination. In some embodiments, O-glycans are removedfrom glycoproteins (e.g., glycoproteins) by non-reductive betaelimination.

In some embodiments, O-linked glycans are removed from a glycoprotein(e.g., a glycoprotein) preparation by incubating the preparation in asolution that includes alkaline tetrahydroborate. In some embodiments,tetradeuterioborate is used, e.g., to incorporate a deuterium label tofacilitate detection of O-linked glycans. In various exemplary methods,a glycoprotein preparation is incubated in a solution containing 0.8-1.0M NaBH₄ and 0.05-0.1 M NaOH at 42-45° C. for 2-24 hours. A reaction toremove O-linked glycans can be terminated by the addition of acid (e.g.,1.0 M HCl).

In some embodiments, O-linked glycans are removed from a glycoproteinpreparation by incubating the preparation in a solution that includesNaOH. In various exemplary methods, a glycoprotein is incubated in asolution containing 50-200 mM NaOH at 27-45° C. for 2-48 hours. Areaction can be terminated by the addition of acid.

In some embodiments, O-linked glycans are removed from a glycoproteinpreparation by incubating the preparation in a solution that includesNH₄OH. In various exemplary methods, a glycoprotein is incubated in asolution containing 25-28% NH₄OH at 45-60° C. for 2-40 hours. Thereaction can be terminated by removing the NH₄OH under vacuum. In someembodiments, the solution includes ammonium carbonate (e.g., at asaturating concentration). In some embodiments, the NH₄OH-treatedpreparation is treated with acid (e.g., boric acid).

In some embodiments, O-linked glycans are removed from a glycoproteinpreparation by incubating the preparation in an aqueous solution thatincludes ethylamine (e.g., ethylamine at about 70%) or methylamine(e.g., methylamine at about 40%), for about 4-24 hours.

In some embodiments, an O-linked glycan preparation is obtained from aglycoprotein population from which N-linked glycans have been removed.

Labeling Glycans

In some embodiments, labels can be associated with glycans before orafter release from a glycoprotein. N-linked glycans or O-linked glycans(e.g., N-glycans that have been removed from a glycoprotein population)can be associated with one or more detectable labels. Detectable labelsare typically associated with the reducing ends of glycans. In someembodiments, detectable labels are fluorescent moieties. Exemplaryfluorophores that can be used in accordance with the present disclosureinclude, but are not limited to, 2-aminobenzoic acid (2AA),2-aminobenzamide (2AB), and/or 2-aminopurine (2AP). In general,fluorophores for use in accordance with the present disclosure arecharacterized by having reactivity with the reducing end of anoligosaccharide and/or monosaccharide under conditions that do notdamage and/or destroy the glycan. In some embodiments, fluorescentmoieties are attached to reducing ends directly. For example, directattachment can be accomplished by direct conjugation by reductiveamination. In some embodiments, fluorescent moieties are attached toreducing ends indirectly. For example, indirect attachment can beaccomplished by a reactive linker arm.

In some embodiments, detectable labels comprise radioactive moieties orisotopically-labelled molecules. Exemplary radioactive moieties that canbe used in accordance with the present disclosure include, but are notlimited to, tritium (³H), deuterium (²H), and/or ³⁵S. Typically, suchmoieties are directly attached to or otherwise associated with thefluorophore. To give but one example of a radioactive fluorophore, 2APcan be modified such that all hydrogens are deuterated.

Release of Glycans

The present disclosure provides improved methods of determiningglycosylation patterns of glycoproteins. Such methods can involvesubjecting a glycan population to one or more exoglycosidases andanalyzing the structure and/or composition of the digestion products. Insome embodiments, exoglycosidases used in accordance with the presentdisclosure recognize and cleave only one particular type of glycosidiclinkage. In some embodiments, exoglycosidases used in accordance withthe present disclosure recognize and cleave more than one particulartype of glycosidic linkage. Among the exoglycosidases which may beuseful for the present invention are α-galactosidases, β-galactosidases;hexosaminidases, mannosidases; and combinations thereof.

Exoglycosidases

Exoglycosidases are enzymes which cleave terminal glycosidic bonds fromthe non-reducing end of glycans. They are typically highly specific toparticular monosaccharide linkages and anomericity (α/β). In someembodiments, neighboring branching patterns can affect exoglycosidasespecificity. Exoglycosidase treatment usually results in glycans ofstandard antennary linkages being cleaved down to the pentasaccharidecore (M3N2) containing 3 mannose and 2 GlcNAc residues. However,unusually-modified species (e.g., antennary or core fucosylated species,high-mannose and hybrid glycans, lactosamine-extended glycans, sulfatedglycans, phosphorylated glycans, etc.) are resistant to exoglycosidasetreatment and can be chromatographically resolved and quantifiedrelative to the M3N2 pentasaccharide.

Exemplary exoglycosidases that can be used in accordance with thepresent disclosure include, but are not limited to, sialidase,galactosidase, hexosaminidase, fucosidase, and mannosidase.Exoglycosidases can be obtained from any source, including commercialsources or by isolation and/or purification from a cellular source(e.g., bacteria, yeast, plant, etc.).

In some embodiments, exoglycosidases (e.g., sialidases, galactosidases,hexosaminidases, fucosidases, and mannosidases) can be divided intomultiple categories or “subsets.” In some embodiments, the differentsubsets display different abilities to cleave different types oflinkages. Table 1 presents some exemplary exoglycosidases, their linkagespecificities, and the organism from which each is derived. One ofordinary skill in the art will appreciate that this is an exemplary, nota comprehensive, list of exoglycosidases, and that any exoglycosidasehaving any linkage specificity may be used in accordance with thepresent disclosure.

TABLE 1 Exoglycosidases Enzyme class EC #* Activity Organism α-Sialidase3.2.1.18 α-2/3,6,8 (usually not linkage- Arthrobacter ureafaciensspecific) Vibrio cholerae Clostridium perfringens α-2,3 (NeuAc fromoligosaccharides) Salmonella typhimurium Streptococcus pneumonia α-2/3,6(NeuAc from complex) Clostridium perfringens β-Galactosidase 3.2.1.23β-1/3,4,6 Gal linkages Bovine testis Xanthamonas species Streptococcusspecies E. coli β-1/4,6 Gal linkages Jack bean β-1,4 Gal linkageStreptococcus pneumonia β-1,3-Gal linkage E. coli Xanthomonas speciesβ-1/3,6-Gal linkages Xanthomonas species E. coli β-Hexosaminidase3.2.1.52 β-1/2,3,4,6 hexosamines Streptococcus plicatus 3.2.1.30Streptococcus pneumonia Bacteroides Jack bean α-Fucosidase 3.2.1.51α-1-3,4-Fuc (usually de-glycosylate Xanthomonas 3.2.1.111 Lewisstructure) Almond meal α-1/2,3,4,6-Fuc (usually has broad Bovine kidneyspecificity) C. meningosepticum α-1,6-Fuc E. coli α-1,2-Fuc Xanthomonasα-Mannosidase 3.2.1.24 α-1/2,3,6-Man Jack bean α-1/2,3-Man Xanthomonasmanihotis α-1,6-Man (typically a core Xanthomonas species mannosidase)α-1,2-Man Aspergillus saitoi β-Mannosidase 3.2.1.25 α-1,4-Man Helixpomatia “EC #” refers to Enzyme Commission registration number

According to the present disclosure, a glycan population can be digestedwith any exoglycosidase or any set of exoglycosidases. In general,exoglycosidase reactions take place under conditions that are compatiblewith enzyme activity. For example, pH, temperature, reaction solutioncomponents and concentration (e.g., salt, detergent, etc.), and lengthof reaction time can be optimized in order to achieve a desired level ofexoglycosidase activity. See, e.g., WO 2008/130926, the contents ofwhich are herein incorporated by reference.

Analysis of Glycan Structure and Activity

In general, methods in accordance with the disclosure comprisesubjecting a glycan preparation to analysis to determine whetherglycoproteins in the preparation include an N-acetylhexosamine glycanstructure. In some embodiments, the analysis comprises comparing thestructure and/or function of glycans in one glycoprotein preparationfrom one source to structure and/or function of glycans in at least oneother glycoprotein preparation from another source. In some embodiments,the analysis comprises comparing the structure and/or function ofglycans in one or more of the samples to structure and/or function ofglycans in a reference sample.

Structure and composition of glycans can be analyzed by any availablemethod. In some embodiments, glycan structure and composition areanalyzed by chromatographic methods, mass spectrometry (MS) methods,chromatographic methods followed by MS, electrophoretic methods,electrophoretic methods followed by MS, nuclear magnetic resonance (NMR)methods, and combinations thereof.

In some embodiments, glycan structure and composition can be analyzed bychromatographic methods, including but not limited to, liquidchromatography (LC), high performance liquid chromatography (HPLC),ultra performance liquid chromatography (UPLC), thin layerchromatography (TLC), amide column chromatography, and combinationsthereof.

In some embodiments, glycan structure and composition can be analyzed bymass spectrometry (MS) and related methods, including but not limitedto, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorptionionisation mass spectrometry (MALDI-MS), Fourier transform massspectrometry (FTMS), ion mobility separation with mass spectrometry(IMS-MS), electron transfer dissociation (ETD-MS), and combinationsthereof.

In some embodiments, glycan structure and composition can be analyzed byelectrophoretic methods, including but not limited to, capillaryelectrophoresis (CE), CE-MS, gel electrophoresis, agarose gelelectrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) followed by Western blotting using antibodiesthat recognize specific glycan structures, and combinations thereof.

In some embodiments, glycan structure and composition can be analyzed bynuclear magnetic resonance (NMR) and related methods, including but notlimited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR),correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), totalcorrelated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantumcoherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence(HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR(ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), andcombinations thereof.

In some embodiments, techniques described herein may be combined withone or more other technologies for the detection, analysis, and orisolation of glycans or glycoproteins. For example, in certainembodiments, glycans are analyzed in accordance with the presentdisclosure using one or more available methods (to give but a fewexamples, see Anumula, Anal. Biochem. 350(1):1, 2006; Klein et al.,Anal. Biochem., 179:162, 1989; and/or Townsend, R. R. CarbohydrateAnalysis” High Performance Liquid Chromatography and CapillaryElectrophoresis., Ed. Z. El Rassi, pp 181-209, 1995, each of which isincorporated herein by reference in its entirety). For example, in someembodiments, glycans are characterized using one or more ofchromatographic methods, electrophoretic methods, nuclear magneticresonance methods, and combinations thereof. Exemplary such methodsinclude, for example, NMR, mass spectrometry, liquid chromatography,2-dimensional chromatography, SDS-PAGE, antibody staining, lectinstaining, monosaccharide quantitation, capillary electrophoresis,fluorophore-assisted carbohydrate electrophoresis (FACE), micellarelectrokinetic chromatography (MEKC), exoglycosidase or endoglycosidasetreatments, and combinations thereof. Those of ordinary skill in the artwill be aware of other techniques that can be used to characterizeglycans together with the methods described herein.

In some embodiments, methods described herein allow for detection ofglycan species (such as an N-acetylhexosamine glycan (e.g., anN-acetylglucosamine and/or N-acetylgalactosamine glycan) that arepresent at low levels within a population of glycans. For example, thepresent methods allow for detection of glycan species that are presentat levels less than 10%, less than 5%, less than 4%, less than 3%, lessthan 2%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%,less than 0.25%, less than 0.1%, less than 0.075%, less than 0.05%, lessthan 0.025%, or less than 0.01% within a population of glycans.

In some embodiments, methods described herein allow for detection ofparticular structures (e.g., an N-acetylhexosamine glycan (e.g., anN-acetylglucosamine and/or N-acetylgalactosamine glycan)) that arepresent at low levels within a population of glycans. For example, thepresent methods allow for detection of particular structures that arepresent at levels less than 10%, less than 5%, less than 4%, less than3%, less than 2%, less than 1.5%, less than 1%, less than 0.75%, lessthan 0.5%, less than 0.25%, less than 0.1%, less than 0.075%, less than0.05%, less than 0.025%, or less than 0.01% within a population ofglycans.

In some embodiments, methods described herein allow for detection ofrelative levels of individual glycan species within a population ofglycans. For example, the area under each peak of a liquid chromatographcan be measured and expressed as a percentage of the total. Such ananalysis provides a relative percent amount of each glycan specieswithin a population of glycans. In another example, relative levels ofindividual glycan species are determined from areas of peaks in a 1D-NMRexperiment, or from volumes of cross peaks from a 1H-15HSQC spectrum(e.g., with correction based on responses from standards), or byrelative quantitation by comparing the same peak across samples.

In some embodiments, a biological activity of a glycoprotein preparation(e.g., a glycosylated antibody preparation) is assessed. Biologicalactivity of the glycoprotein preparation can be analyzed by anyavailable method. In some embodiments, a binding activity of aglycoprotein is assessed (e.g., binding to a receptor). In someembodiments, a therapeutic activity of a glycoprotein is assessed (e.g.,an activity of a glycoprotein in decreasing severity or symptom of adisease or condition, or in delaying appearance of a symptom of adisease or condition). In some embodiments, a pharmacologic activity ofa glycoprotein is assessed (e.g., bioavailability, pharmacokinetics,pharmacodynamics). For methods of analyzing bioavailability,pharmacokinetics, and pharmacodynamics of glycoprotein therapeutics,see, e.g., Weiner et al., J Pharm Biomed Anal. 15(5):571-9, 1997;Srinivas et al., J. Pharm. Sci. 85(1):1-4, 1996; and Srinivas et al.,Pharm. Res. 14(7):911-6, 1997.

As would be understood to one of skill in the art, the particularbiological activity or therapeutic activity that can be tested will varydepending on the particular glycoprotein.

The potential adverse activity or toxicity (e.g., propensity to causehypertension, allergic reactions, thrombotic events, seizures, or otheradverse events) of glycoprotein preparations can be analyzed by anyavailable method. In some embodiments, immunogenicity of a glycoproteinpreparation is assessed, e.g., by determining whether the preparationelicits an antibody response in a subject.

In various embodiments, biological activity, therapeutic activity, etc.,of a glycoprotein preparation that includes an N-acetylhexosamine glycanis compared to a glycoprotein preparation that does not include orincludes at background levels an N-acetylhexosamine glycan. In variousembodiments, biological activity, therapeutic activity, etc., of aglycoprotein preparation having N-acetylhexosamine glycans is comparedto a glycoprotein preparation having a different level ofN-acetylhexosamine glycans.

Applications

Methods of the present disclosure can be utilized to analyze glycansfrom glycoproteins in any of a variety of states including, forinstance, free glycans, glycoproteins (e.g., glycopeptides, glycolipids,proteoglycans, etc.), cell-associated glycans (e.g., nucleus-,cytoplasm-, cell-membrane-associated glycans, etc.); glycans associatedwith cellular, extracellular, intracellular, and/or subcellularcomponents (e.g., proteins); glycans in extracellular space (e.g., cellculture medium), etc.

Methods of the present disclosure may be used in one or more stages ofprocess development for the production of a therapeutic or othercommercially relevant glycoprotein. For example, the methods describedherein can be used to evaluate a production parameter or parametersusing to produce a glycoprotein preparation, to compare glycoproteinpreparations produced by different production parameters, and todetermine and/or select a production parameter or parameters for aglycoprotein preparation such that a particular glycan structure can beobtained upon production of a glycoprotein preparation. A productionparameter as used herein is a parameter or element in a productionprocess. Production parameters that can be selected include, e.g., thecell or cell line used to produce the glycoprotein preparation, theculture medium, culture process or bioreactor variables (e.g., batch,fed-batch, or perfusion), purification process and formulation of aglycoprotein preparation. Exemplary production parameters include: 1)the types of host; 2) genetics of the host; 3) media type; 4)fermentation platform; 5) purification steps; and 6) formulation.

The present disclosure can also be utilized to monitor the extent and/ortype of glycosylation occurring in a particular cell culture (e.g., theextent of N-acetylhexosamine glycans in glycoprotein preparationproduced in the cell culture), thereby allowing adjustment or possiblytermination of the culture in order, for example, to achieve aparticular desired glycosylation pattern or to avoid development of aparticular undesired glycosylation pattern.

The present disclosure can also be utilized to assess glycosylationcharacteristics of cells or cell lines (e.g., CHO cell lines) that arebeing considered for production of a particular desired glycoprotein(for example, even before the cells or cell lines have been engineeredto produce the glycoprotein, or to produce the glycoprotein at acommercially relevant level).

For example, where the target glycoprotein is a therapeuticglycoprotein, for example having undergone regulatory review in one ormore countries, it will often be desirable to monitor cultures to assessthe likelihood that they will generate a product with a glycosylationpattern as close to the established glycosylation pattern of thepharmaceutical product as possible (e.g., having a degree ofN-acetylhexosamine glycan content which is close to that of thepharmaceutical product), e.g., whether or not it is being produced byexactly the same route. As used herein, “close” refers to aglycosylation pattern having at least about a 75%, 80%, 85%, 90%, 95%,98%, or 99% correlation to the established glycosylation pattern of thepharmaceutical product. In such embodiments, samples of the productionculture are typically taken at multiple time points and are comparedwith an established standard or with a control culture in order toassess relative glycosylation.

For example, in some embodiments, methods for monitoring production of aglycoprotein may comprise steps of (i) during production of aglycoprotein, removing at least first and second glycan-containingsamples from the production system; (ii) subjecting each of the firstand second glycan-containing samples to an analysis to determine whethera particular modification is present (e.g., an N-acetylhexosamineglycan); and (iii) comparing the products obtained from the firstglycan-containing sample with those obtained from the secondglycan-containing sample so that differences are determined andtherefore progress of glycoprotein production is monitored. In someembodiments, the production system comprises CHO cells.

Whether or not monitoring production of a particular target protein forquality control purposes, the present disclosure may be utilized, forexample, to monitor glycosylation at particular stages of development,or under particular growth conditions.

In some embodiments, methods described herein can be used tocharacterize, modulate and/or control or compare the quality oftherapeutic products. To give but one example, the present methodologiescan be used to assess glycosylation in cells producing a therapeuticprotein product. Particularly given that glycosylation can often affectthe activity, bioavailability, or other characteristics of a therapeuticprotein product, methods for assessing cellular glycosylation duringproduction of such a therapeutic protein product are particularlydesirable. Among other things, the present disclosure can facilitatereal time analysis of glycosylation in production systems fortherapeutic proteins, and hence, modulation of the glycosylation may beachieved.

In some embodiments, the glycoprotein preparation is a glycosylatedantibody preparation. The term “antibody” refers to a protein thatincludes at least one immunoglobulin variable domain (variable region)or immunoglobulin variable domain (variable region) sequence. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH or HV), and a light (L) chain variable region(abbreviated herein as VL or LV). In another example, an antibodyincludes two heavy (H) chain variable regions and two light (L) chainvariable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies as well as complete antibodies. The term“antigen-binding fragment” of a full length antibody refers to one ormore fragments of a full-length antibody that retain the ability tospecifically bind to a target of interest.

An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM(as well as subtypes thereof). Antibodies may be from any source, butprimate (human and non-human primate) and primatized are preferred.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. In IgGs, the heavy chainconstant region includes three immunoglobulin domains, CH1, CH2 and CH3.The light chain constant region includes a CL domain. The variableregion of the heavy and light chains contains a binding domain thatinteracts with an antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.

In one embodiment, the glycosylated antibody has one or more regionsthat are human or effectively human. For example, one or more of thevariable regions can be human or effectively human. For example, one ormore of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1,LC CDR2, and/or LC CDR3. Each of the light chain (LC) and/or heavy chain(HC) CDRs can be human. HC CDR3 can be human. One or more of theframework regions can be human, e.g., FR1, FR2, FR3, and/or FR4 of theHC and/or LC. For example, the Fc region can be human. In oneembodiment, all the framework regions are human.

Representative glycoprotein products include, for example, theglycosylated antibodies provided in Table 2:

TABLE 2 Exemplary Commercially Available Glycoprotein Products ProteinProduct Reference Listed Drug Alefacept; recombinant, dimeric Amevive ®fusion protein LFA3-Ig Bevacizumab Avastin ™ Tositumomab BEXXAR ®Alemtuzumab Campath ® acritumomab; technetium-99 labeled CEA-Scan ®crotalidae polyvalent immune Fab, ovine CroFab ™ digoxin immune Fab[ovine] DigiFab ™ Etanercept ENBREL ® Cetuximab Erbitux ™ TrastuzumabHerceptin ® Somatotropin Humatrope ® Adalimumab HUMIRA ™ RanibizumabLUCENTIS ® gemtuzumab ozogamicin Mylotarg ™ Fanolesomab NeutroSpec ™(formerly LeuTech ®) immunoglobulin intravenous Octagam ® abatacept,fully human soluble fusion protein Orencia ™ muromomab-CD3 OrthocloneOKT3 ® Infliximab REMICADE ® Abciximab ReoPro ™ Rituximab Rituxan ™Basiliximab Simulect ® Eculizumab SOLIRIS (R) Palivizumab; recombinantlyproduced, Synagis ™ humanized mAb Natalizumab TYSABRI ® human immuneglobulin intravenous Venoglobulin-S ® 5% and 10% solutions Omalizumab;recombinant DNA-derived Xolair ® humanized monoclonal antibody targetingimmunoglobulin-E Daclizumab Zenapax ® ibritumomab tiuxetan Zevalin ™

In some embodiments, the disclosure provides methods in which glycansfrom glycoproteins from different sources or samples are compared withone another. In some such examples, multiple samples from the samesource (e.g., from the same CHO cell source) are obtained over time, sothat changes in glycosylation patterns (and particularly in cell surfaceglycosylation patterns) (e.g., changes in the presence or extent ofN-acetylhexosamine glycans) are monitored. In some embodiments, one ofthe samples is a historical sample or a record of a historical sample.In some embodiments, one of the samples is a reference sample.

In some embodiments, the disclosure provides methods in which glycansfrom glycoproteins expressed by different cell sources are compared withone another. In some embodiments, one or more of the compared cellsources are CHO cells.

In some embodiments, glycans from different cell culture samplesprepared under different production parameters (e.g., cell type, culturetype (e.g., continuous feed vs. batch feed, etc.), culture conditions(e.g., type of media, presence or concentration of particular componentof particular medium(a), osmolarity, pH, temperature, timing or degreeof shift in one or more components such as osmolarity, pH, temperature,etc.), culture time, isolation steps, etc.) but are otherwise identical,are compared, so that effects of the selected production parameter onglycosylation are determined. In certain embodiments, glycans fromdifferent cell culture samples prepared under conditions that differ ina single selected production parameter are compared so that effects ofthe single selected production parameter on glycosylation patterns(e.g., the absence, presence, or extent of N-acetylhexosamine glycans)are determined. Among other applications, therefore, use of techniquesas described herein may facilitate determination of the effects ofparticular production parameters on glycosylation patterns in cells.

In some embodiments, glycans from different batches of a glycoprotein,whether prepared by the same method or by different methods, and whetherprepared simultaneously or separately, are compared. In suchembodiments, the present disclosure facilitates quality control of aglycoprotein preparation. Alternatively or additionally, some suchembodiments facilitate monitoring of progress of a particular cultureproducing a glycoprotein (e.g., when samples are removed from theculture at different time points and are analyzed and compared to oneanother). In some examples, multiple samples from the same source areobtained over time, so that changes in glycosylation patterns aremonitored. In some embodiments, glycan-containing samples are removed atabout 30 second, about 1 minute, about 2 minute, about 5 minute, about10 minute, about 30 minute, about 1 hour, about 2 hour, about 3 hour,about 4 hour, about 5 hour, about 10 hour, about 12 hour, or about 18hour intervals, or at even longer intervals. In some embodiments,glycan-containing samples are removed at irregular intervals. In someembodiments, glycan-containing samples are removed at 5 hour intervals.

In some embodiments, methods in accordance with the disclosure may beused to monitor the glycosylation pattern of glycoproteins during thecourse of their production by cells. For example, production of aglycoprotein (e.g., commercial production) may involve steps of (1)culturing cells that produce the glycoprotein, (2) obtaining samples atregular or irregular intervals during the culturing, and (3) analyzingthe glycosylation pattern of produced glycoprotein(s) in obtainedsample(s). In some embodiments, such methods may comprise a step ofcomparing the glycosylation patterns of produced glycoprotein(s) inobtained samples to one another. In some embodiments, such methods maycomprise a step of comparing glycosylation patterns of producedglycoprotein(s) in obtained sample(s) to the glycosylation pattern of areference sample.

In some embodiments, methods in accordance with the disclosure may beused to monitor the glycosylation pattern of glycoproteins over thecourse of storage. For example, the method can include obtaining samplesat regular or irregular intervals during the storage of a glycoproteinpreparation, and (3) analyzing the glycosylation pattern ofglycoprotein(s) in obtained sample(s). In some embodiments, such methodsmay comprise a step of comparing the glycosylation patterns of theglycoprotein(s) in obtained samples to one another. In some embodiments,such methods may comprise a step of comparing glycosylation patterns ofthe glycoprotein(s) in obtained sample(s) to the glycosylation patternof a reference sample.

In any of these embodiments, features of the glycan analysis can berecorded, for example in a quality control record. As indicated above,in some embodiments, a comparison is with a historical record of a prioror standard batch and/or with a reference sample of glycoprotein.

In some embodiments, glycans from different batches of a particularglycoprotein, whether prepared by the same method or by differentmethods, and whether prepared simultaneously or separately, are comparedto one another and/or to a reference sample. In some embodiments,batch-to-batch comparison may comprise the steps of (i) providing afirst glycan preparation from a first batch of the glycoprotein; (ii)providing a second glycan preparation from a second batch of theglycoprotein; (iii) subjecting each of the first and second glycanpreparations to analysis procedure; and (iv) comparing the results ofthe analysis obtained from the first glycan preparation with thecleavage products obtained from the second preparation so thatconsistency of the two batches is assessed. In some embodiments, glycanpreparations can be provided by removing at least one glycan from atleast one glycoprotein from a batch and, optionally, isolating removedglycans. In some embodiments, glycan preparations may be labeled asdescribed herein (e.g., fluorescently and/or radioactively; e.g., priorto and/or after isolation).

In some embodiments, the present disclosure facilitates quality controlof a glycoprotein preparation. Features of the glycan analysis can berecorded, for example in a quality control record. As indicated above,in some embodiments, a comparison is with a historical record of a prioror standard batch of glycoprotein. In some embodiments, a comparison iswith a reference glycoprotein sample.

In certain embodiments, the present disclosure may be utilized instudies to modify the glycosylation characteristics of a cell, forexample to establish a cell line and/or culture conditions with one ormore desirable glycosylation characteristics, e.g., a cell line thatproduces glycoproteins having, having a certain amount or lacking anN-acetylhexosamine glycan. Such a cell line and/or culture conditionscan then be utilized, if desired, for production of a particular targetglycoprotein for which such glycosylation characteristic(s) is/areexpected to be beneficial. In particular embodiments, the cell is a CHOcell.

According to the present disclosure, techniques described herein can beused to detect desirable or undesirable glycans, for example to detector quantify the presence of one or more contaminants in a glycoproteinproduct, or to detect or quantify the presence of one or more active ordesired species.

In certain embodiments, methods described herein facilitate detection ofglycans that are present at very low levels in a source (e.g., abiological sample, glycan preparation, etc.). In such embodiments, it ispossible to detect and/or optionally quantify the levels of glycans thatare present at levels less than about 10%, 5%, 4%, 3%, 2%, 1.5%, 1%,0.75%, 0.5%, 0.25%, 0.1%, 0.075%, 0.05%, 0.025%, or 0.01% within apopulation of glycans. In some embodiments, it is possible to detectand/or optionally quantify the levels of glycans comprising between 0.1%and 5%, e.g., between 0.1% and 2%, e.g., between 0.1% and 1% of a glycanpreparation.

In some embodiments, methods described herein allow for detection ofrelative levels of individual glycan species within a population ofglycans. For example, the area under each peak of a liquid chromatographcan be measured and expressed as a percentage of the total. Such ananalysis provides a relative percent amount of each glycan specieswithin a population of glycans. In some embodiments, methods describedherein allow for the manufacture of a glycoprotein, e.g., a glycoproteincontaining an N-acetylhexosamine glycan, e.g., N-acetylglucosamine orN-acetylgalactosamine. For example, the manufacture of a glycoprotein(e.g., commercial production) may involve steps of (1) culturing cellsthat produce the glycoprotein, (2) obtaining samples of the glycoprotein(e.g., at regular or irregular intervals during the culturing, and/or atthe end of a culturing process) and (3) analyzing the glycosylationpattern of produced glycoprotein(s) in obtained sample(s) for thepresence, absence and/or amount of an N-acetylhexosamine glycan, e.g.,N-acetylglucosamine or N-acetylgalactosamine. In some embodiments, suchmethods may comprise a step of comparing the glycosylation patterns ofproduced glycoprotein(s) in obtained samples to a reference, e.g.,comparing the presence, absence and/or amount of an N-acetylhexosamineglycan, e.g., N-acetylglucosamine or N-acetylgalactosamine in theproduced glycoprotein, to a reference, such as a pharmaceuticalspecification, e.g., a pharmaceutical specification for the producedglycoprotein for the presence, absence and/or amount of anN-acetylhexosamine glycan, e.g., N-acetylglucosamine orN-acetylgalactosamine. In some embodiments, the methods may comprise astep of comparing glycosylation patterns of produced glycoprotein(s) inobtained sample(s) to the glycosylation pattern of a reference sample.

In some embodiments, the method comprises further processing of theglycoprotein, e.g., the further processing can include combining theglycoprotein preparation with a second component, e.g., an excipient orbuffer. In one embodiment, the further processing can include one ormore of: formulating the glycoprotein preparation; processing theglycoprotein n preparation into a drug product; combining theglycoprotein preparation with a second component, e.g., an excipient orbuffer; changing the concentration of the glycoprotein in thepreparation; lyophilizing the glycoprotein preparation; combining afirst and second aliquot of the glycoprotein to provide a third, larger,aliquot; dividing the glycoprotein preparation into smaller aliquots;disposing the glycoprotein preparation into a container, e.g., a gas orliquid tight container; packaging the glycoprotein preparation;associating a container comprising the glycoprotein preparation with alabel; shipping or moving the glycoprotein preparation to a differentlocation.

The present disclosure will be more specifically illustrated withreference to the following examples. However, it should be understoodthat the present disclosure is not limited by these examples in anymanner.

One of skill in the art may readily envision various other combinationswithin the scope of the present invention, considering the example withreference to the specification herein provided.

EXAMPLES Example 1

The truncated N-linked glycan identified was unexpected based on thebiosynthetic glycosylation pathway as it is understood. In general, aglycoprotein proceeds through the standard glycosylation cascaderesulting in a number of potential N-linked glycan species, all of whichcontain at least a core structure containing 2 GlcNAc moieties and 3mannose residues. This species identified consists of only a singleN-acetylhexosamine linked to Asn297 in the Fc domain of the antibody.The species was identified though mass spectrometric analysis of thetryptic digest of a monoclonal antibody. A species having a mass 203 Dahigher than the expected mass of the non-glycosylated peptide wasobserved as the [M+2H]²⁺ ion with an m/z of 696.8 (which is the mass ofthe peptide with one HexNAc). MS/MS interrogation of this speciesrevealed the peptide contains a hexosamine residue linked to the singleasparagine residue that comprises the peptide (EEQYNSTYR) (FIG. 2).

This species was also found in several commercial antibodies at varyinglevels (FIG. 3). Peptides were derived from each of the commerciallyavailable antibody products through the use of enzymatic cleavage (i.e.trypsin). Peptides were reduced and alkylated and analyzed by LC-MS withMS/MS used for peptide sequencing. The amino acid sequence from peptidesequencing was used to determine the identity of the peptide.Glycopeptides were observed as a mass shift relative to thenon-glycosylated peptide. The relative abundance of HexNAc-glycopeptidein various commercially available antibody products was calculated basedon the abundance of the G0F (fucosylated, but no galactose residuesbiantennery glycan), G1F (fucosylated and one galactose residuebiantennery glycan), G2F (fucosylated and two galactose residues,biantennery glycan), and the aglycosyl glycopeptides.

The presence of this species was not confined to a particular class ofantibody as it was identified in both IgG1 and IgG2 therapeutics.

Procedure Used to Analyze the Glycan Species:

Peptides were derived from the drug substance through the use ofenzymatic cleavage (i.e. trypsin). Peptides were reduced and alkylatedand analyzed by LC-MS with MS/MS used for peptide sequencing. The aminoacid sequence from peptide sequencing was used to determine the identityof the peptide. Glycopeptides were observed as a mass shift relative tothe non-glycosylated peptide.

This data illustrates the presence of an N-linked N-acetylhexosamine inthe glycosylated monoclonal antibody. FIG. 2 shows the fluorescencechromatogram of a fraction of glycans derived from the monoclonalantibody.

Extensions and Alternatives

While the methods has been particularly shown and described withreference to specific illustrative embodiments, it should be understoodthat various changes in form and detail may be made without departingfrom the spirit and scope of the present disclosure. Therefore, allembodiments that come within the scope and spirit of the methods, andequivalents thereto, are intended to be claimed. The claims,descriptions and diagrams of the methods, systems, and assays of thepresent disclosure should not be read as limited to the described orderof elements unless stated to that effect.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols. The section headings used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed in any way. While the methods have been described inconjunction with various embodiments and examples, it is not intendedthat the methods be limited to such embodiments or examples. On thecontrary, the methods encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

1-35. (canceled)
 36. A method for manufacturing an antibody preparation,the method comprising: culturing cells to produce a recombinant antibodypreparation; determining the absence, presence or amount of an N-linkedglycan consisting of a single N-acetylglucosamine residue in theproduced preparation; and processing the antibody preparation as apharmaceutical product based upon the determination.
 37. The method ofclaim 1, further comprising comparing the determined absence, presenceor amount of N-linked glycans consisting of a single N-acetylglucosamineresidue to a reference value for the antibody preparation for theabsence, presence or amount of N-linked glycans consisting of a singleN-acetylglucosamine residue.
 38. The method of claim 1, wherein thedetermining step comprises the use of a method for identifying orquantifying N-linked glycans consisting of a single N-acetylglucosamineresidue selected from the group consisting of: chromatographic methods,mass spectrometry (MS) methods, electrophoretic methods, nuclearmagnetic resonance (NMR) methods, monosaccharide analysis, fluorescencemethods, UV-VIS absorbance, enzymatic methods, use of a detectionmolecule, and combinations thereof.
 39. The method of claim 1, whereinthe cells are cultured in a bioreactor.
 40. The method of claim 1,wherein the processing step comprises disposing the antibody preparationinto a container and the container is a gas or liquid tight container.41. The method of claim 1, wherein the cells are CHO cells.
 42. Themethod of claim 1, wherein the processing step comprises one or more of:formulating the antibody preparation; processing the antibodypreparation into a drug product; combining the antibody preparation witha second component; lyophilizing the antibody preparation.
 43. Themethod of claim 1, wherein the processing step comprises formulating theantibody preparation.
 44. The method of claim 1, wherein the processingstep comprises processing the antibody preparation into a drug product.45. The method of claim 1, wherein the processing step compriseslyophilizing the antibody preparation.
 46. The method of claim 1,wherein the processing step comprises one or more of: formulating theantibody preparation; processing the antibody preparation into a drugproduct; combining the antibody preparation with a second component;changing the concentration of the antibody in the preparation;lyophilizing the antibody preparation; combining a first and secondaliquot of the antibody to provide a third, larger, aliquot; dividingthe antibody preparation into smaller aliquots; disposing the antibodypreparation into a container; packaging the antibody preparation;associating a container comprising the antibody preparation with alabel; shipping or moving the antibody preparation to a differentlocation.
 47. The method of claim 1, wherein the antibody is atherapeutic antibody.
 48. The method of claim 1, wherein the cells aremammalian cells.
 49. The method of claim 1, wherein the antibodypreparation comprises an N-linked glycan consisting of a singleN-acetylglucosamine residue.
 50. The method of claim 14, wherein theantibody preparation comprises an N-linked glycan consisting of a singleN-acetylglucosamine residue linked to the Fc domain of the antibody. 51.The method of claim 14, wherein the antibody preparation comprises anN-linked glycan consisting of a single N-acetylglucosamine residuelinked to Asn297 of the Fc domain of the antibody.
 52. A method formanufacturing a fusion protein preparation, the method comprising:culturing cells to produce a recombinant fusion protein preparation;determining the absence, presence or amount of an N-linked glycanconsisting of a single N-acetylglucosamine residue in the producedpreparation; and processing the fusion protein preparation as apharmaceutical product based upon the determination.
 53. The method ofclaim 17, further comprising comparing the determined absence, presenceor amount of N-linked glycans consisting of a single N-acetylglucosamineresidue to a reference value for the fusion protein preparation for theabsence, presence or amount of N-linked glycans consisting of a singleN-acetylglucosamine residue.
 54. The method of claim 17, wherein thedetermining step comprises the use of a method for identifying orquantifying N-linked glycans consisting of a single N-acetylglucosamineresidue selected from the group consisting of: chromatographic methods,mass spectrometry (MS) methods, electrophoretic methods, nuclearmagnetic resonance (NMR) methods, monosaccharide analysis, fluorescencemethods, UV-VIS absorbance, enzymatic methods, use of a detectionmolecule, and combinations thereof.
 55. The method of claim 17, whereinthe cells are cultured in a bioreactor.
 56. The method of claim 17,wherein the processing step comprises disposing the fusion proteinpreparation into a container and the container is a gas or liquid tightcontainer.
 57. The method of claim 17, wherein the cells are CHO cells.58. The method of claim 17, wherein the processing step comprises one ormore of: formulating the fusion protein preparation; processing thefusion protein preparation into a drug product; combining the fusionprotein preparation with a second component; lyophilizing the fusionprotein preparation.
 59. The method of claim 17, wherein the processingstep comprises formulating the fusion protein preparation.
 60. Themethod of claim 17, wherein the processing step comprises processing thefusion protein preparation into a drug product.
 61. The method of claim17, wherein the processing step comprises lyophilizing the fusionprotein preparation.
 62. The method of claim 17, wherein the processingstep comprises one or more of: formulating the fusion proteinpreparation; processing the fusion protein preparation into a drugproduct; combining the fusion protein preparation with a secondcomponent; changing the concentration of the fusion protein in thepreparation; lyophilizing the fusion protein preparation; combining afirst and second aliquot of the fusion protein to provide a third,larger, aliquot; dividing the fusion protein preparation into smalleraliquots; disposing the fusion protein preparation into a container;packaging the fusion protein preparation; associating a containercomprising the fusion protein preparation with a label; shipping ormoving the fusion protein preparation to a different location.
 63. Themethod of claim 17, wherein the fusion protein is a therapeutic fusionprotein.
 64. The method of claim 17, wherein the cells are mammaliancells.
 65. The method of claim 17, wherein the fusion proteinpreparation comprises an N-linked glycan consisting of a singleN-acetylglucosamine residue.
 66. The method of claim 30, wherein thefusion protein preparation comprises an N-linked glycan consisting of asingle N-acetylglucosamine residue linked to an Fc domain of the fusionprotein.
 67. The method of claim 30, wherein the fusion proteinpreparation comprises an N-linked glycan consisting of a singleN-acetylglucosamine residue linked to Asn297 of an Fc domain of thefusion protein.